Project Summary Targeting tumor-associated carbohydrate antigens (TACAs) for anticancer vaccines is exceptionally attractive because many TACAs are highly expressed on multiple types of cancer cells with no or negligible expression on normal cells. While passive immunity utilizing anti-TACA monoclonal antibodies has achieved clinical success, developing a TACA based immunogen to elicit effective anti-cancer immunity has been extremely difficult due to the notoriously low immunogenicity of TACAs. To address this challenge, exciting preliminary results have been obtained showing that TACA-based vaccines can significantly reduce cancer induced death by delivering a prototypical TACA, the Tn antigen, using virus like particle bacteriophage Q?. In this proposal, new ground in TACA based cancer vaccine design will continue to be broken by engineering Q? to generate powerful anti-cancer immune responses. Building on the exciting preliminary results, in Aim 1, an important tumor associated glycopeptide antigen human MUC-1 bearing Tn glycan will be targeted. Using Q? as the carrier, super-high titers of IgG antibodies were elicited against MUC1-Tn, which significantly reduced tumor growth in mice, even in transgenic mice tolerant to human MUC1. Besides Tn and tumor associated MUC1 glycopeptides, other TACAs including GD2 and SSEA-3 will be investigated as vaccine targets to reduce the risk of tumor escape from immune surveillance and to kill purported cancer stem cells, a possible cause for resistance to traditional treatments such as chemotherapy. In addition, guided by the crystal structure of Q?, Q? mutants will be developed to reduce undesirable anti-Q? antibodies and further boost desired anti- TACA responses. Cytotoxic T cells can also kill cancer cells. In Aim 2, Q? will be engineered to deliver cytotoxic T cell epitopes and built-in adjuvants in addition to generating anti-TACA IgG antibodies. The comprehensive antibody and cytotoxic T cell immune responses induced by Q?-TACA-cytotoxic T cell epitope conjugate should provide superior protection to immunized host against tumor development. To lay the groundwork for future translation, in Aim 3, the vaccine efficacy in treating canine cancer patients will be established. Canines can naturally develop cancer, which are clinically relevant large animal models for human diseases due to their high similarities to human cancer. This will be the first of its kind trial of such TACA based vaccine constructs in canine patients. Overall impacts: This project will establish a Q? vaccine platform vastly superior to currently available carriers to deliver both TACAs and cytotoxic T cell epitopes, which will elicit long-lasting anti-TACA IgG antibodies and cytotoxic T cells for cancer treatment. Deeper understanding of the connections between structural features of Q?-TACA conjugates and anti-tumor immunity will exert a sustained impact on cancer vaccine design and is essential for successful TACA-based anti-cancer vaccines.